Molecular chaperones are a promising therapeutic target in the treatment of neurological diseases of protein misfolding. This is because of their ability to interact with client proteins that become misfolded and aggregate-prone. One of these clients, the microtubule associated protein tau, abnormally aggregates in numerous neurodegenerative diseases termed tauopathies. Some chaperone proteins have shown auspicious results as therapeutic targets in models of tauopathy. However, this study proposes to add to the evidence for Hsp70-mediated interventions for the discovery of compounds that reduce tau levels. The chaperone activity of Hsp70 is powered by the hydrolysis of ATP. Moreover, many pharmacological compounds that modulate the rate of ATP hydrolysis of Hsp70 have been shown to modulate the levels of tau: One compound that was shown to inhibit the ATPase activity of Hsp70 is methylene blue (MB). MB has been shown to reduce tau levels in various models of tauopathy and has been shown to rescue cognition. These data suggest that Hsp70 ATPase modulation may be a viable tool for the development of anti-tau therapies. However, there is vast literature describing the many other interactions and effects of MB. As a result, the current data create a stronger case for MB as a promising therapeutic candidate than for Hsp70's ATPase as a therapeutic target. Strong evidence for Hsp70 as a therapeutic target will allow drug developers to create new compounds that may hold the cure for tauopathies. Thus, we have created new genetics-based tools with which to study this question in order to collect data that will either support or refute the hypothesis. We have created single amino acid mutants of Hsp70 and tested their ATPase and refolding activity as well as their effects on tau levels. One mutation, E175S, on Hsp70 reduces its ATPase activity and refolding activity. Furthermore, it reduces tau levels in cell models of tauopathy. Thus, we have replicated the previous in vitro data that used Hsp70 inhibitor compounds. The objective of this study is to test the hypothesis that the inhibition of Hsp70 ATPase activity degrades tau through the cellular degradation pathways. To achieve this, our design consists of the following specific aims: 1) We will block and activate the proteasome, macroautophagy and chaperone-assisted autophagy pathways in the presence of Hsp70E175S overexpression; 2) We will determine if inhibiting the ATPase activity of Hsp70 reduces tau in vivo and evaluate the therapeutic potential of this strategy. PUBLIC HEALTH RELEVANCE: The protein tau is very important in the normal activities of brain cells. Due to unknown reasons tau loses its normal function and becomes toxic in a large number of neurological diseases. The number of people suffering from these diseases is large and is projected to increase dramatically due to an increase in the aging population. Unfortunately, the current treatments available for most of these diseases only reduce symptoms for a brief period of time. As a result, the discovery of therapies that could halt, prevent or significantly slow the repercussions of tau toxicity is an urgent goal in the scientific field.